Chronic infections by the opportunistic pathogen Pseudomonas aeruginosa are the major cause of morbidity in patients suffering from cystic fibrosis (CF). In the CF lung, P. aeruginosa faces and successfully responds to numerous stress and starvation conditions, such as oxidative stress, low iron concentrations and low oxygen and nutrient availability within bacterial biofilms. One such response is the induction of virulence gene expression. Many of these virulence genes are controlled by quorum sensing (QS), a mechanism that allows cell-density-dependent regulation of gene expression. Our long-term goal is to elucidate the regulatory mechanisms that control virulence gene expression in P. aeruginosa. Our specific hypothesis is that stress and starvation induction of QS gene expression is controlled by the stringent response. The stringent response is a global regulatory mechanism that reprograms gene expression in response to slow growth. Importantly, P. aeruginosa mutants deficient in eliciting the stringent response through the central signaling molecule ppGpp show reduced virulence and expression of several QS genes. We further hypothesize that the observed inhibition of QS by the macrolide antibiotic azithromycin is mediated by repression of the stringent response. Our specific aims are (1) to define the stringent-response-dependent transcriptome of P. aeruginosa, (2) to investigate the role of the stringent response in stress and starvation-dependent QS gene expression, (3) to investigate the growth rate dependence of ppGpp production and QS gene expression, and (4) to investigate the role of the stringent response in the inhibition of QS virulence gene expression by azithromycin. Our proposed research will provide important insights into the function of macrolides as anti-quorum sensing drugs. It will help elucidate the molecular components necessary for P. aeruginosa survival in the CF lung and uncover novel antivirulence strategies. PUBLIC HEALTH RELEVANCE: Chronic infections by the bacterial pathogen Pseudomonas aeruginosa are the major cause of morbidity in patients suffering from cystic fibrosis. P. aeruginosa resists and responds to the generally very hostile conditions faced in the CF lung, including nutrient deprivation and oxidative stress. One such response is the increased production of virulence factors. Many of these factors are controlled by quorum sensing, a mechanisms that allows regulation of gene expression according to population size. Our proposed research will investigate a link between bacterial virulence gene expression and starvation responses. It will help understand the components necessary for P. aeruginosa survival in the CF lung and uncover novel antivirulence strategies.